Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/16—Primer sets for multiplex assays

Definitions

• the present invention relates to Chlamydia trachomatis and Neisseria gonorrhoeae .
• the invention relates to polynucleotides and methods for amplifying and detecting Chlamydia trachomatis and Neisseria gonorrhoeae.
• Chlamydia trachomatis C. trachomatis or CT
• Neisseria gonorrhoeae N. gonorrhoeae or NG
• CT causes venereal lymphogranuloma, various inflammatory pathologies of the male and female urogenital systems, and trachoma, a chronic disease that affects 500 million people and can lead to blindness.
• CT-induced urethritis and cervicitis may led to a variety of chronic inflammations, such as, e.g., vaginitis, salpingitis and pelvic inflammation which may result in sterility and extrauterine pregnancy.
• the new born from infected mothers may contract pulmonary and/or ocular infections during delivery.
• N. gonorrhoeae the pathogen of gonorrhoea, manifests itself as a purulent inflammation and swelling of the urethra in males. These symptoms occur in 90% of cases of infection. If left untreated, the infection can ascend and after several weeks produce symptoms of prostatitis. In women, no or only slight symptoms occur in 50% of cases of infection. The infection primarily affects the cervix, but also the urethra. In 10 to 15% of women, the infection spreads to the fallopian tubes and can also lead to sterility. Since the course of the infections is often asymptomatic, many carriers contribute unknowingly to the spread of the disease.
• antibody titres may be undetectable in acute infections (false negative), may persist in uninfected individuals with a past history of infection (false positive), may yield a false-positive indication due to the presence of cross-reacting species (e.g., respiratory infection by different Chlamydia species), or may not develop at all (false negative) depending on other factors (Ngeow, 1996, Ann Acad Med Singapore 25:300; Black et al., 1991, J Clin Microbiol 29:1312). For these reasons, serology alone is inadequate for the diagnosis of CT and NG infections.
• Bacterial infections may also be diagnosed by the detection of nucleic acid sequences particular to the infectious organism.
• a diagnostic assay may be specific for an entire genus, more than one genus, a species or subspecies, auxotype, serovar (serotype), strain or other subset of organisms. This selectivity may be exploited in the development of simple reliable diagnostic tests for C. trachomatis and N. gonorrhoeae species of bacterial pathogens.
• the present invention provides polynucleotide reagents useful for the detection of Chlamydia trachomatis and/or Neisseria gonorrhoeae .
• the present invention provides primers and probes for nucleic acid amplification and detection procedures, which specifically and sensitively detect various subspecies or serotypes and auxotypes of C. trachomatis and/or N. gonorrhoeae.
• Two or more of the polynucleotide reagents provided herein as SEQ ID NOs: 1-12 can be combined in a composition, such as SEQ ID NO: 1 and one or more of SEQ ID NOs: 4-7 or such as SEQ ID NO: 2 and one or more of SEQ ID NOs: 4-7.
• two more of the polynucleotide reagents provided herein can be combined such as one or more of SEQ ID NOs: 8 and one of SEQ ID NOs: 9-12, or SEQ ID NO: 9 and one of SEQ ID NOs: 8 and 10-12.
• compositions comprise a first polynucleotide having a sequence selected from SEQ ID NOs: 1-3, a second polynucleotide having the sequence set forth in SEQ ID NO: 4, a third polynucleotide having the sequence set forth in SEQ ID NO: 8, and a fourth polynucleotide having the sequence set forth in SEQ ID NO: 9.
• the primer sets herein provided comprise two primers, and are useful for the amplification of target sequences, e.g., in PCR.
• the primers designated SEQ ID NOs: 1-3 and SEQ ID NO: 4 specifically amplify C. trachomatis
• the primers designated SEQ ID NO: 8 and SEQ ID NO: 9 amplify serotypes and/or auxotypes of N. gonorrhoeae .
• sequences identified herein as SEQ ID NOs: 5-7 are probe sequences useful for detecting amplified Chlamydia trachomatis , e.g., when Chlamydia trachomatis is amplified by primers having sequences identified by SEQ ID NOs: 1-3 and 4.
• sequences identified herein as SEQ ID NOs: 10-12 are useful for detecting amplified N. gonorrhoeae , e.g., when N. gonorrhoeae is amplified by primers having sequences identified by SEQ ID NOs: 8 and 9.
• the primer and probe set comprises two primer sequences and one probe sequence. More preferably, the primer and probe set are used for PCR.
• primer/probe sets that can be employed to amplify and detect C. trachomatis include primer/probe sets 1-9, which are set forth below.
• specific primer/probe sets that can be employed to amplify and detect N. gonorrhoeae include primer/probe sets 10-12 which are set forth below.
• the method for amplifying C. trachomatis and/or N. gonorrhoeae will generally comprise (a) forming a reaction mixture comprising nucleic acid amplification reagents, at least one polynucleotide of the invention, or composition, or primer set, or primer/probe set as mentioned above, and a test sample potentially containing at least one target sequence and (b) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the target sequence.
• the method for detecting C. trachomatis and/or N. gonorrhoeae will generally comprise (a) forming a reaction mixture comprising nucleic acid amplification reagents, at least one polynucleotide of the invention, or composition, or primer set, or primer/probe set as mentioned above, and a test sample potentially containing at least one target sequence; (b) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the target sequence; (c) hybridizing the probe to the nucleic acid sequence complementary to the target sequence, so as to form a hybrid comprising the probe and the nucleic acid sequence complementary to the target sequence; and (d) detecting the hybrid as an indication of the presence of C. trachomatis and/or N. gonorrhoeae in the test sample.
• step (b) can be repeated multiple times to increase the number of target sequence copies.
• both C. trachomatis and N. gonorrhoeae can be detected in a single reaction mixture using a combination of the primer/probe sets, especially when a probe having SEQ ID NO: 5, 6, or 7 carries a label that produces a signal distinct from the signal generated by a probe having SEQ ID NO: 10, 11, or 12, and one of each set is contacted to a test sample amplified with SEQ ID NOs: 1-3 and 4, in combination with SEQ ID NOs: 8 and 9.
• kits useful for amplifying and /or detecting C. trachomatis and/or N. gonorrhoeae .
• the kits comprise one or more suitable containers containing one or more primer sets or primer/probe sets or primer/probe combinations according to the present invention, an enzyme having polymerase activity and deoxynucleotide triphosphates. At least one sequence preferably bears a label.
• a control target polynucleotide and a control polynucleotide probe are included in any of the methods or kits provided herein.
• the primer set preferably is complementary to the target sequence as well as the control target, whereas the target probe is preferably substantially complementary only to the target polynucleotide sequence and the control probe is preferably substantially complementary only to the control target.
• Another aspect of the invention provides isolated polynucleotides having a nucleotide sequence consisting essentially of, and preferably consisting of, the nucleotide sequences set forth in SEQ ID NOs: 2-12.
• the present invention provides polynucleotides that can specifically hybridise with a nucleic acid sequence, or complement thereof, of Chlamydia trachomatis ( C. trachomatis or CT) and further provides other polynucleotides that can specifically hybridise with a nucleic acid sequence, or complement thereof, of Neisseria gonorrhoeae ( N. gonorrhoeae or NG). These polynucleotides can be used to amplify Chlamydia trachomatis and N. gonorrhoeae , and to specifically detect the presence of each organism to the exclusion of the other.
• sixteen strains of Chlamydia trachomatis and 57 auxotypes and serovars of N. gonorrhoeae are known, and the method suitably and specifically detects all known strains.
• specifically hybridise refers to the ability of a nucleic acid to bind detectably and specifically to a second nucleic acid.
• Stringent conditions that can be used to achieve specific hybridisation are known in the art.
• target sequence or “target nucleic acid sequence” as used herein means a nucleic acid sequence of CT or NG, or complement thereof, that is amplified, detected, or both amplified and detected using one or more of the polynucleotides herein provided. Additionally, while the term target sequence sometimes refers to a double stranded nucleic acid sequence, those skilled in the art will recognize that the target sequence can also be single stranded. In cases where the target is double stranded, polynucleotide primer sequences of the present invention preferably will amplify both strands of the target sequence. A target sequence may be selected that is more or less specific for a particular organism.
• the target sequence may be specific to an entire genus, to more than one genus, to a species or subspecies, serogroup, auxotype, serotype, strain, isolate or other subset of organisms.
• the polynucleotide sequences of the present invention are selected for their ability to specifically hybridize with a range of subspecies, or serotypes or auxotypes, of CT and NG.
• test sample means a sample taken from an organism or biological fluid that is suspected of containing or potentially contains a CT or NG target sequence.
• the test sample can be taken from any biological source, such as for example, tissue, blood, saliva, sputa, mucus, sweat, urine, urethral swabs, cervical swabs, urogenital or anal swabs, conjunctival swabs, ocular lens fluid, cerebral spinal fluid, milk, ascites fluid, synovial fluid, peritoneal fluid, amniotic fluid, fermentation broths, cell cultures, chemical reaction mixtures and the like.
• test sample can be used (i) directly as obtained from the source or (ii) following a pre-treatment to modify the character of the sample.
• the test sample can be pre-treated prior to use by, for example, preparing plasma or serum from blood, disrupting cells or viral particles, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, distilling liquids, concentrating liquids, inactivating interfering components, adding reagents, purifying nucleic acids, and the like.
• label means a molecule or moiety having a property or characteristic which is capable of detection and, optionally, of quantitation.
• a label can be directly detectable, as with, for example (and without limitation), radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent microparticles and the like; or a label may be indirectly detectable, as with, for example, specific binding members. It will be understood that directly detectable labels may require additional components such as, for example, substrates, triggering reagents, quenching moieties, light, and the like to enable detection and/or quantitation of the label. When indirectly detectable labels are used, they are typically used in combination with a “conjugate”.
• a conjugate is typically a specific binding member that has been attached or coupled to a directly detectable label. Coupling chemistries for synthesizing a conjugate are well known in the art and can include, for example, any chemical means and/or physical means that does not destroy the specific binding property of the specific binding member or the detectable property of the label.
• “specific binding member” means a member of a binding pair, i.e., two different molecules where one of the molecules through, for example, chemical or physical means specifically binds to the other molecule.
• binding pairs include, but are not intended to be limited to, avidin and biotin; haptens and antibodies specific for haptens; complementary nucleotide sequences; enzyme cofactors or substrates and enzymes; and the like.
• a polynucleotide in the context of the present invention, is a nucleic acid polymer of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), modified RNA or DNA, or RNA or DNA mimetics (such as, without limitation PNAs), and derivatives thereof, and homologues thereof.
• RNA ribonucleic acid
• DNA deoxyribonucleic acid
• DNA mimetics such as, without limitation PNAs
• derivatives thereof such as, without limitation PNAs
• polynucleotides include polymers composed of naturally occurring nucleobases, sugars and covalent intemucleoside (backbone) linkages as well as polymers having non-naturally-occurring portions that function similarly.
• modified or substituted nucleic acid polymers are well known in the art and for the purposes of the present invention, are referred to as “analogues.”
• polynucleotides are preferably modified or unmodified polymers of deoxyribonucleic acid or ribonucleic acid.
• modified backbones include those retaining a phosphorus atom in the backbone, such as phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, as well as those no longer having a phosphorus atom, such as backbones formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
• modified nucleic acid polymers may contain one or more modified sugar moieties.
• sugar moieties may be modified by substitution at the 2′ position with a 2-methoxyethoxy (2-MOE) group (see, for example, Martin et al., (1995) Helv. Chim. Acta , 78:486-504).
• the present invention also contemplates analogues that are RNA or DNA mimetics, in which both the sugar and the internucleoside linkage of the nucleotide units are replaced with novel groups.
• the base units are maintained for hybridisation with the target sequence.
• An example of such a mimetic which has been shown to have excellent hybridisation properties, is a peptide nucleic acid (PNA) (Nielsen et al., (1991) Science , 254:1497-1500; International Patent Application WO 92/20702, herein incorporated by reference).
• PNA peptide nucleic acid
• the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, for example an aminoethylglycine backbone.
• the nucleobases are retained and are bound directly or indirectly to the aza-nitrogen atoms of the amide portion of the backbone.
• Contemplated polynucleotides of the invention further include derivatives wherein the nucleic acid molecule has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring nucleotide, for example with a moiety that functions as a label, as described herein.
• the present invention further encompasses homologues of the polynucleotides having nucleic acid sequences set forth in SEQ ID NOs: 1-12.
• Homologues are nucleic acids having at least one alteration in the primary sequence set forth in any one of SEQ ID NOs: 1-12, that does not destroy the ability of the polynucleotide to specifically hybridise with a target sequence, as described above. Accordingly, a primary sequence can be altered, for example, by the insertion, addition, deletion or substitution of one or more of the nucleotides of, for example, SEQ ID NOs: 1-12.
• homologues that are fragments of a sequence disclosed in SEQ ID NOs: 1-12 may have a consecutive sequence of at least about 7, 10, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23 or more nucleotides of the nucleic acid sequences of SEQ ID NO: 1-12, and will retain the ability to specifically hybridise with a target sequence, as described above.
• the homologues will have a nucleic acid sequence having at least about 50%, 60%, 70%, 80%, 85%, 90% or 95% nucleic acid sequence identity with a nucleic acid sequence set forth in SEQ ID NOs: 1-12.
• Identity with respect to such sequences is defined herein as the percentage of nucleotides in the candidate sequence that are identical with the known polynucleotides after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Terminal (5′ or 3′) or internal deletions, extensions or insertions into the nucleotide sequence shall not be construed as affecting identity.
• the polynucleotides of the present invention thus comprise primers and probes that specifically hybridize to a target sequence of the invention, for example the nucleic acid molecules having any one of the nucleic acid sequences set forth in SEQ ID NOs: 1-12, including analogues and/or derivatives of said nucleic acid sequences, and homologues thereof, that can specifically hybridise with a target sequence of the invention.
• polynucleotides of the invention can be used as primers and/or probes to amplify or detect Chlamydia trachomatis or N. gonorrhoeae.
• hybridizing polynucleotides are defined further as being novel and non-obvious over any prior art polynucleotide including that which hybridizes under appropriate stringency conditions, a target sequence according to the present invention.
• the polynucleotides according to the present invention can be prepared by conventional techniques well known to those skilled in the art.
• the polynucleotides can be prepared using conventional solid-phase synthesis using commercially available equipment, such as that available from Applied Biosystems USA Inc. (Foster City, Calif.), DuPont, (Wilmington, Del.), or Milligen (Bedford, Mass.).
• Modified polynucleotides, such as phosphorothioates and alkylated derivatives can also be readily prepared by similar methods known in the art. See, for example, U.S. Pat. Nos. 5,464,746; 5,424,414; and 4,948,882.
• the polynucleotides according to the present invention can be employed directly as probes for the detection, or quantitation, or both, of CT and/or NG nucleic acids in a test sample.
• the test sample is contacted with at least one of the polynucleotides of the present invention under suitable hybridisation conditions and the hybridization between the target sequence and at least one of the polynucleotides is then detected by methods well-known in the art.
• the polynucleotides of the present invention may incorporate one or more detectable labels.
• Detectable labels are molecules or moieties having a property or characteristic that can be detected directly or indirectly and are chosen such that the ability of the polynucleotide to hybridise with its target sequence is not adversely affected.
• Methods of labeling nucleic acid sequences are well known in the art (see, for example, Ausubel et al., (1997 & updates) Current Protocols in Molecular Biology , Wiley & Sons, New York).
• Detection labels have the same definition as “labels” previously defined and “capture labels” are typically used to separate extension products, and probes associated with any such products, from other amplification reactants. Specific binding members (as previously defined) are well suited for this purpose. Also, probes used according to this method may be blocked at their 3′ ends so that they are not extended under hybridization conditions. Methods for preventing extension of a probe are well known and are a matter of choice for one skilled in the art. Typically, adding a phosphate group to the 3′ end of the probe will suffice for purposes of blocking extension of the probe.
• primer sequences optionally can be labeled with either a capture label or a detection label.
• the probe sequence is used to hybridize with the sequence generated by the primer sequence, and typically hybridizes with a sequence that does not include the primer sequence.
• the probe sequence can also labeled with either a capture label or a detection label with the caveat that when the primer is labeled with a capture label, the probe is labeled with a detection label, and vice versa.
• the differential labels i.e., capture and detection labels
• detection is performed according to the protocols used by the commercially available Abbott LCx® instrumentation (Abbott Laboratories; Abbott Park, Ill.).
• the polynucleotides according to the present invention are also suitable for use as capture probes in sandwich-type assays.
• Capture probes and sandwich hybridisation assays are well known in the art. Briefly, the polynucleotide capture probe is attached to a solid support and brought into contact with a test sample under suitable hybridisation conditions such that a probe:target hybrid is formed between the capture probe and any target nucleic acid present in the test sample. After one or more appropriate washing steps, the probe:target hybrid is detected, usually by means of a second “disclosure” probe or by a specific antibody that recognises the hybrid molecule.
• the use of the CT and/or NG polynucleotides of the invention as a capture or a disclosure probe, or both, in such sandwich hybridisation assays is thus considered to be within the scope of the present invention.
• the present invention also contemplates the use of the polynucleotides in modified nucleic acid hybridisation assays.
• U.S. Pat. No. 5,627,030 discloses a method to amplify the detection signal in a nucleic acid hybridisation assay.
• a first polynucleotide probe sequence is hybridised under suitable conditions to a target sequence, the probe:target hybrid is subsequently immunocaptured and immobilised.
• a second polynucleotide probe which contains many repeating sequence units is then hybridised to the probe component of the probe:target hybrid. Detection is achieved by hybridisation of many labeled nucleic acid sequence probes, one to each of the repeating sequence units present in the second probe.
• the polynucleotides of the invention can be used as primers or probes to amplify and/or detect CT or NG in a test sample.
• the primer/probe sets provided herein comprise two primers and at least one probe. These primer/probe sets can be employed according to nucleic acid amplification techniques. Hence, the primers in any particular primer/probe set can be employed to amplify a target sequence. In most cases, the probe hybridizes to the copies of the target sequence generated by one of the primers and generally facilitates detecting any copies of the target sequence generated during the course of the amplification reaction.
• primer/probe sets can be employed according to nucleic acid amplification procedures to specifically and sensitively detect either CT or NG, or both CT and NG when the appropriate primers and probes are combined. It is contemplated that the individual primers and probes of the primer/probe sets provided herein may alternatively be used in combination with primers and/or probes other than those described in the primer/probe sets provided herein.
• Amplification procedures are well-known in the art and include, but are not limited to, polymerase chain reaction (PCR), TMA, rolling circle amplification, nucleic acid sequence based amplification (NASBA), and strand displacement amplification (SDA).
• PCR polymerase chain reaction
• TMA rolling circle amplification
• NASBA nucleic acid sequence based amplification
• SDA strand displacement amplification
• the primers may need to be modified, for example, for SDA the primer comprises additional nucleotides near its 5′ end that constitute a recognition site for a restriction endonuclease.
• NASBA the primer comprises additional nucleotides near the 5′ end that constitute an RNA polymerase promoter. Polynucleotides thus modified are considered to be within the scope of the present invention.
• the primers should be selected such that the likelihood of forming 3′ duplexes is minimised, and such that the melting temperatures (T M ) are sufficiently similar to optimise annealing to the target sequence and minimise the amount of non-specific annealing.
• T M melting temperatures
• the polynucleotides according to the present invention are provided in combinations that can be used as primers in amplification reactions to specifically amplify target nucleic acid sequences.
• polynucleotides having the nucleic acid sequences as set forth in SEQ ID NOs: 1, 2, or 3 used in combination with the nucleic acid sequences as set forth in SEQ ID NO: 4, which are provided with polynucleotides having the nucleic acid sequences as set forth in SEQ ID NOs: 8 and 9.
• these primer combinations are used to specifically amplify CT and/or NG nucleic acid sequences, if present in a test sample.
• Primers included in the primer/probe sets 1-9 can be used to prime synthesis and amplification of copies of a CT target sequence in the case of SEQ ID NOs: 1, 2, or 3 and 4; and copies of a NG target sequence in the case of primer/probe sets 10-12 and SEQ ID NOs: 8 and 9.
• the remaining SEQ ID NOs can specifically hybridize with the amplification products of the primer sequences found in the same primer/probe set.
• primer/probe set 10 is specific for NG insofar as SEQ ID NOs: 8 and 9 prime synthesis of the NG target sequence and SEQ ID NO: 10 hybridizes with the amplification products produced by SEQ ID NOs: 8 and 9.
• the amplification method of the present invention generally comprises (a) forming a reaction mixture comprising nucleic acid amplification reagents, at least one primer/probe set of the present invention, and a test sample suspected of containing a at least one target sequence and (b) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the target sequence.
• Step (b) of the above methods can be repeated any suitable number of times (prior to step (c) in the detection method), e.g., by thermal cycling the reaction mixture between 10 and 100 times, typically between about 20 and about 60 times, more typically between about 25 and about 45 times.
• Nucleic acid amplification reagents include reagents which are well known and may include, but are not limited to, an enzyme having at least polymerase activity, enzyme cofactors such as magnesium or manganese; salts; nicotinamide adenine dinucleotide (NAD); and deoxynucleotide triphosphates (dNTPs) such as for example deoxyadenine triphosphate, deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythymine triphosphate.
• enzyme cofactors such as magnesium or manganese
• salts such as for example deoxyadenine triphosphate, deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythymine triphosphate.
• dNTPs deoxynucleotide triphosphates
• Amplification conditions are conditions that generally promote annealing and extension of one or more nucleic acid sequences. It is well known that such annealing is dependent in a rather predictable manner on several parameters, including temperature, ionic strength, sequence length, complementarity, and G:C content of the sequences. For example, lowering the temperature in the environment of complementary nucleic acid sequences promotes annealing. For any given set of sequences, melt temperature, or Tm, can be estimated by any of several known methods. Typically, diagnostic applications utilize hybridization temperatures that are about 10° C. (e.g., 2° C. to 18° C.) below the melt temperature.
• Ionic strength or “salt” concentration also impacts the melt temperature, since small cations tend to stabilize the formation of duplexes by negating the negative charge on the phosphodiester backbone.
• Typical salt concentrations depend on the nature and valency of the cation but are readily understood by those skilled in the art.
• high G:C content and increased sequence length are also known to stabilize duplex formation because G:C pairings involve 3 hydrogen bonds where A:T pairs have just two, and because longer sequences have more hydrogen bonds holding the sequences together.
• a high G:C content and longer sequence lengths impact the hybridization conditions by elevating the melt temperature.
• the G:C content and length will be known and can be accounted for in determining precisely what hybridization conditions will encompass. Since ionic strength is typically optimized for enzymatic activity, the only parameter left to vary is the temperature. Generally, the hybridization temperature is selected close to or at the Tm of the primers or probe. Thus, obtaining suitable hybridization conditions for a particular primer/probe set is well within ordinary skill of one practicing this art.
• the primer sequences (SEQ ID NOs: 1-4, 8 and 9) of any particular primer/probe set can, by themselves or with additional polynucleotides, be used as amplification primers according to nucleic acid amplification procedures well known in the art.
• Such reactions include, but are not intended to be limited to, the polymerase chain reaction (PCR) described in U.S. Pat. Nos. 4,683,195 and 4,683,202, the ligase chain reaction (LCR) described in EP-A-320 308, and gap LCR (GLCR) described in U.S. Pat. No. 5,427,930.
• PCR polymerase chain reaction
• LCR ligase chain reaction
• GLCR gap LCR
• the detection method generally comprises (a) forming a reaction mixture comprising nucleic acid amplification reagents, at least one primer/probe set of the present invention, and a test sample suspected of containing at least one target sequence; (b) subjecting the mixture to amplification conditions to generate at least one copy of a nucleic acid sequence complementary to the target sequence; (c) hybridizing the probe to the nucleic acid sequence complementary to the target sequence, so as to form a hybrid comprising the probe and the nucleic acid sequence complementary to the target sequence; and (d) detecting the hybrid as an indication of the presence of the target sequence (CT and/or NG and/or a control sequence) in the sample.
• CT and/or NG and/or a control sequence detecting the hybrid as an indication of the presence of the target sequence in the sample.
• amplicons produced by amplification of target nucleic acid sequences using the polynucleotides of the present invention can be detected by a variety of methods known in the art.
• one or more of the primers used in the amplification reactions may be labeled such that an amplicon can be directly detected by conventional techniques subsequent to the amplification reaction.
• a probe consisting of a labeled version of one of the primers used in the amplification reaction, or a third polynucleotide distinct from the primer sequences that has been labeled and is complementary to a region of the amplified sequence can be added after the amplification reaction is complete. The mixture is then submitted to appropriate hybridisation and wash conditions and the label is detected by conventional methods.
• the amplification product produced as above can be detected during or subsequently to the amplification of the target sequence.
• Methods for detecting the amplification of a target sequence during amplification are outlined above, and described, for example, in U.S. Pat. No. 5,210,015.
• Gel electrophoresis can be employed to detect the products of an amplification reaction after its completion.
• amplification products are hybridized to probes, then separated from other reaction components and detected using microparticles and labeled probes.
• the present invention thus includes the use of the polynucleotides in a method to specifically amplify and detect target nucleic acid sequences in a test sample in a single tube format.
• This may be achieved, for example, by including in the reaction vessel an intercalating dye such as SYBR Green or an antibody that specifically detects the amplified nucleic acid sequence.
• an intercalating dye such as SYBR Green or an antibody that specifically detects the amplified nucleic acid sequence.
• a third polynucleotide distinct from the primer sequences, which is complementary to a region of the amplified sequence may be included in the reaction, as when a primer/probe set of the invention is used.
• the polynucleotide probe For use in an assay as described above, in which both amplification with polynucleotide primers and detection of target sequences using a polynucleotide probe occur concurrently in a single unopened reaction vessel, the polynucleotide probe needs to possess certain properties. For example, since the probe will be present during the amplification reaction, it should not interfere with the progress of this reaction and should also be stable under the reaction conditions. In addition, for real-time monitoring of reactions, the probe should be capable of binding its target sequence under the conditions of the amplification reaction and to emit a signal only upon binding this target sequence. Examples of probe molecules that are particularly well-suited to this type of procedure include molecular beacon probes and TaqMan® probes.
• TaqMan® probes are dual-labeled fluorogenic nucleic acid probes composed of a polynucleotide complementary to the target sequence that is labeled at the 5′ terminus with a fluorophore and at the 3′ terminus with a quencher. TaqMan® probes are typically used as real-time probes in amplification reactions. In the free probe, the close proximity of the fluorophore and the quencher ensures that the fluorophore is internally quenched.
• the probe is cleaved by the 5′ nuclease activity of the polymerase and the fluorophore is released.
• the released fluorophore can then fluoresce and thus produces a detectable signal.
• molecular beacon probes are well known in the art, for example, see U.S. Pat. Nos: 6,150,097; 5,925,517 and 6,103,476. Basically, molecular beacons are polynucleotide probes capable of forming a stem-loop (hairpin) structure. The loop is a single-stranded structure containing sequences complementary to the target sequence, whereas the stem typically is unrelated to the target sequence and self-hybridises to form a double-stranded region. Nucleotides that are both complementary to the target sequence and that can self-hybridise may also form part of the stem region.
• Attached to one arm of the stem is a fluorophore moiety and to the other arm a quencher moiety.
• the fluorophore and the quencher are in close proximity and the energy emitted by the fluorophore is thus absorbed by the quencher and given off as heat, resulting in internal quenching of the fluorophore.
• the fluorophore and the quencher Upon binding of the polynucleotide to its target sequence, the fluorophore and the quencher become spatially separated and the fluorophore can fluoresce producing a detectable signal.
• the primers and probes of the present invention are selected such that the probe stably (e.g., less than 5% of probe bound to amplicon is displaced over 24 hours under the hybridization conditions for the probe to the amplicon) binds to the amplicon when the reaction is cooled from a denaturation temperature, e.g., 90° C. to 96° C., to a temperature below the Tm for the binding of the probe with the amplicon.
• a denaturation temperature e.g. 90° C. to 96° C.
• the present invention further contemplates the use of polynucleotides of the invention as linear probes in conjunction with a fluorophore and a high efficiency quencher, such as the Black Hole Quenchers (BHQTM; Biosearch Technologies, Inc., Novato, Calif.).
• a fluorophore and a high efficiency quencher such as the Black Hole Quenchers (BHQTM; Biosearch Technologies, Inc., Novato, Calif.).
• BHQTM Black Hole Quenchers
• the high quenching efficiency and lack of native fluorescence of the BHQTM dyes allows “random-coil” quenching to occur in linear probes labeled at one terminus with a fluorophore and at the other with a BHQTM dye thus ensuring that the fluorophore does not fluoresce when the probe is in solution.
• the probe Upon binding its target sequence, the probe stretches out, the fluorophore and quencher are thus spatially separated and the fluorophore fluoresces.
• BHQTM dyes can also be used as the quencher moiety in molecular beacon or TaqMan® probes.
• Suitable fluorophores and quenchers for use with the polynucleotides of the present invention can be readily determined by one skilled in the art (see also, Tyagi et al., Nature Biotechnol ., 16:49-53 (1998); Marras et al., Genet. Anal.: Biomolec. Eng ., 14:151-156 (1999)). Many fluorophores and quenchers are available commercially, for example from Molecular Probes (Eugene, Oreg.) or Biosearch Technologies, Inc. (Novato, Calif.).
• fluorophores examples include, but are not limited to, fluorescein and fluorescein derivatives such as a dihalo-(C 1 to C 8 )dialkoxycarboxyfluorescein, 5-(2′-aminoethyl)aminonaphthalene-1-sulphonic acid (EDANS), coumarin and coumarin derivatives, Lucifer yellow, Texas red, tetramethylrhodamine, tetrachloro-6-carboxyfluoroscein, 5-carboxyrhodamine, cyanine dyes and the like.
• fluorescein and fluorescein derivatives such as a dihalo-(C 1 to C 8 )dialkoxycarboxyfluorescein, 5-(2′-aminoethyl)aminonaphthalene-1-sulphonic acid (EDANS), coumarin and coumarin derivatives, Lucifer yellow, Texas red, tetramethylrhodamine, tetrachloro-6-car
• Quenchers include, but are not limited to, DABCYL, 4′-(4-dimethylaminophenylazo)benzoic acid (DABSYL), 4dimethylaminophenylazophenyl-4-dimethylaminophenylazophenyl-4′-maleimide (DABMI), tetramethylrhodamine, carboxytetramethylrhodamine (TAMRA), BHQTM dyes and the like.
• DABCYL 4′-(4-dimethylaminophenylazo)benzoic acid
• DABMI 4dimethylaminophenylazophenyl-4-dimethylaminophenylazophenyl-4′-maleimide
• TAMRA carboxytetramethylrhodamine
• BHQTM dyes BHQTM dyes and the like.
• the probes are molecular beacon probes.
• certain criteria need to be met for a molecular beacon probe to be successful in monitoring or detecting an amplification reaction.
• the present invention therefore, provides molecular beacon probes that comprise polynucleotides of the present invention together with flanking self-complementary regions.
• the polynucleotides of the present invention may make up the loop region of the molecular beacon, or they may make up the loop region and part of the stem region.
• the self-complementary stem sequences can be unrelated to the target sequence or may contain one or more nucleotides that are complementary to the target sequence.
• polynucleotides having a nucleic acid sequence as set forth in any one of SEQ ID NOs: 7 or 12, or homologues of these polynucleotides, together with appropriate self-complementary flanking sequences are provided as molecular beacon probes.
• the molecular beacon probes have a nucleic acid sequence as set forth in any one of SEQ ID NOs: 7 or 12.
• primers to be used with the molecular beacon probe also requires certain criteria to be met. For example, it is important that there are no areas of complementarity that may cause the molecular beacon to bind to a primer, which would result in a high background signal.
• polynucleotides according to the present invention are further provided in combinations, comprising two primers and at least one probe, that can be used to specifically amplify and detect target nucleic acid sequences in a test sample.
• primer/probe sets are provided for the amplification and detection of target nucleic acid sequences by molecular beacon PCR.
• molecular beacon probes can be used to monitor the progress of an amplification reaction in real time.
• an amplification reaction such as a PCR
• the molecular beacon interacts with its target sequence at the annealing temperature for the probe, and a fluorescent signal is generated.
• the number of target strands produced in the amplification reaction increases, the number of molecular beacons bound to their target increases concomitantly, as does the strength of the fluorescent signal.
• the combinations of two primers and at least one probe, as described above can be used in either end-point amplification and detection assays, in which the strength of the detectable signal is measured at the conclusion of the amplification reaction, or in real-time amplification and detection assays, in which the strength of the detectable signal is monitored throughout the course of the amplification reaction.
• CT primers may be used in conjunction with primers for the amplification of NG to amplify target sequences from either or both organisms in a single amplification reaction.
• the co-amplification reaction, followed by species-specific hybridization reactions, allow for concurrent assessment of a test sample for infection with either CT, or NG, or both organisms.
• both CT and NG can be amplified and/or detected simultaneously in a single reaction mixture using a combination of two primer/probe sets (i.e. one selected from the CT specific primer/probe sets and the other selected from the NG specific primer/probe sets).
• a test sample could be contacted with primer/probe sets 7and 12, or with primer/probe sets 2 and 10, along with amplification reagents to amplify and detect the presence of CT and NG in a test sample.
• each probe must be detectably distinct from each of the other probes.
• the fluorophore moieties of each beacon probe should fluoresce at different wavelengths.
• the polynucleotides according to the present invention can also be used in assays to quantitate the amount of CT and/or NG nucleic acid present in a sample.
• the polynucleotides according to the present invention can be used in a method to specifically amplify, detect and quantitate target nucleic acid sequences in a test sample, which generally comprises the steps of:
• step (b) of the above method can be repeated several times prior to step (c) by thermal cycling the reaction mixture by standard techniques known in the art.
• the standard can consist of a standard curve compiled by amplification and detection of known quantities of CT or NG nucleic acids under the assay conditions.
• an internal standard can be included in the reaction.
• Such internal standards generally comprise a control target nucleic acid sequence and a control polynucleotide probe.
• the internal standard can optionally further include an additional pair of primers. The primary sequence of these control primers may be unrelated to the polynucleotides of the present invention and specific for the control target nucleic acid sequence.
• control target sequence is designed such that it binds at one end with a primer from a first primer/probe set directed to a first target sequence (for example, CT), and binds at the other end with a primer for a second primer/probe set directed to a second target sequence (for example, NG), such that copies will be generated under amplifying conditions.
• first target sequence for example, CT
• second target sequence for example, NG
• a control target nucleic acid sequence is a nucleic acid sequence that:
• control polynucleotide probe for use in quantitation reactions:
• control target nucleic acid and the control probe is not important provided that they both meet the criteria outlined above.
• the amount of target nucleic acid in a test sample can be quantified using “end point” methods or “real time” methods.
• end point methods or “real time” methods.
• the polynucleotides of the present invention can be conventional hybridisation probes, linear BHQTM probes, TaqMan® probes, molecular beacon probes, or combinations or modified versions thereof.
• the polynucleotides are used as molecular beacon probes.
• the present invention also contemplates the provision of any one or more of the polynucleotides of the invention, for example any one of the primer sets or primer/probe sets of the invention together with a control target nucleic acid sequence, which can be amplified by the specified primer pair, and a control polynucleotide probe for the quantitative reactions.
• the present invention further provides for the inclusion of control primers, which specifically amplify the control target nucleic acid sequence, in the quantitative reactions.
• the amplification and/or detection methods in which the polynucleotides according to the present invention can be employed are suitable for adaptation as high-throughput assays. High-throughput assays provide the advantage of processing many samples simultaneously and significantly decrease the time required to screen a large number of samples.
• the present invention therefore, contemplates the use of the polynucleotides of the present invention in high-throughput screening or assays to detect and/or quantitate CT and/or NG nucleic acids in a plurality of test samples.
• reaction components are usually housed in a multi-container carrier or platform, such as a multi-well microtiter plate, which allows a plurality of assay reactions containing different test samples to be monitored in the same assay.
• a multi-container carrier or platform such as a multi-well microtiter plate
• the present invention also contemplates highly automated high-throughput assays to increase the efficiency of the screening or assay process.
• Many high-throughput screening or assay systems are now available commercially, as are automation capabilities for many procedures such as sample and reagent pipetting, liquid dispensing, timed incubations, formatting samples into microarrays, microplate thermocycling and microplate readings in an appropriate detector, resulting in much faster throughput times.
• kits that allows for the detection and/or quantitation of CT and/or NG nucleic acids.
• kits comprise one or more of the polynucleotides of the invention for use as a primer and/or probe.
• the polynucleotides are provided in the kits in combinations for use as primers to specifically amplify CT and/or NG nucleic acids in a test sample.
• the polynucleotides are provided in combinations that comprise the nucleic acid sequences as set forth in SEQ ID NOs: 1 and 4; and/or SEQ ID NOs: 8 and 9.
• the polynucleotides are provided in the kits in combinations comprising at least two primers and at least one probe.
• the polynucleotides are provided in combinations that comprise the nucleic acid sequences as set forth in SEQ ID NOs: 1, 4, and 5; SEQ ID NOs: 2, 4, and 5; SEQ ID NOs: 3, 4, and 5, SEQ ID NOs: 1, 4, and 6; SEQ ID NOs: 2, 4, and 6; SEQ ID NOs: 8, 9 and 10; SEQ ID NOs: 8, 9 and 11;or SEQ ID NOs: 8, 9 and 12.
• Kits comprising combinations of more than one primer/probe set are also envisaged, e.g., without limitation, SEQ ID NOs: 1, 4, 5, 8, 9 and 10; and SEQ ID NOs: 2, 4, 5, 8, 9 and 10.
• Kits for the detection of CT and/or NG nucleic acids may additionally contain a control target nucleic acid and a control polynucleotide probe.
• the kits comprise one of the above combinations of polynucleotides comprising at least two primers and at least one probe, together with a control target nucleic acid sequence, which can be amplified by the specified primer pair, and a control polynucleotide probe.
• the present invention further provides kits that include control primers, which specifically amplify the control target nucleic acid sequence.
• kits can optionally include amplification reagents, reaction components and/or reaction vessels. Typically, at least one sequence bears a label, but detection is possible without this. Thus, one or more of the polynucleotides provided in the kit may have a detectable label incorporated, or the kit may include reagents for labeling the polynucleotides. One or more of the components of the kit may be lyophilised and the kit may further comprise reagents suitable for the reconstitution of the lyophilised components. The kit can additionally contain instructions for use.
• the polynucleotides, methods, and kits of the present invention are useful in clinical or research settings for the detection and/or quantitation of CT and/or NG nucleic acids.
• the polynucleotides can be used in assays to diagnose CT and/or NG infection in a subject, or to monitor the quantity of a CT and/or NG target nucleic acid sequence in a subject infected with CT and/or NG.
• Monitoring the quantity of bacteria in a subject is particularly important in identifying or monitoring response to anti-bacterial therapy.
• nucleotide probe sequences complementary to the bacterial sequences are indicated in capital letters; lower case nucleotides are self-complementary so as to form a stem of a beacon probe under suitable conditions; and in some instances, nucleotides complementary to the bacterial sequences also form part of the self-complementary beacon probe stem.
• Suitable primer probe sets include, but are not limited to:
• SEQ ID NOs: 1 and 4 are used as consensus amplification primers specific for the CT target sequence.
• SEQ ID NO: 6 or 7 is used as consensus internal hybridization probes for the CT amplification product.
• SEQ ID NOs: 8 and 9 are used as consensus amplification primers specific for the NG target sequence and SEQ ID NO: 10 or 12 is used as consensus internal hybridization probes for the NG amplification product.
• Consensus primers were designed to detect all known CT or NG subtypes by oligonucleotide hybridization PCR.
• these primers were SEQ ID NO: 1 and SEQ ID NO: 4 for CT, and SEQ ID NO: 8 and SEQ ID NO: 9 for NG.
• SEQ ID NO: 3 was found to have some advantages relative to SEQ ID NO: 1 for some embodiments of the present invention, which relate to ease of purification of these polynucleotides. However, those embodiments are not illustrated below, and the inventors prefer use of SEQ ID NO: 1 to use of SEQ ID NO: 3.
• Consensus probes were designed to hybridize with the amplified CT or NG target sequence by oligonucleotide hybridization. These probe sequences were modified for use as probes in a molecular beacon assay by the addition of terminal nucleotides to generate self-complementary 5′ and 3′ ends, and of fluorescent (fluo) and quenching moieties at opposite ends of the polynucleotide (see Table 2).
• CT probe sequence of SEQ ID NO: 6 was the basis of the molecular beacon probe of SEQ ID NO: 7
• the NG probe sequence of SEQ ID NO: 10 was the basis of the molecular beacon probe of SEQ ID NO: 12. Where more than one beacon probe was used simultaneously in the same molecular beacon assay, fluorescing moieties with different excitation-emission profiles were used to distinguish between the different probe signals.
• Probe sequences were synthesized using standard oligonucleotide synthesis methodology.
• CT DNA was isolated from CT serotype Ba, Apache-2 strain (ATCC VR-347. Rockville, Md.) using methods known in the art, and used to assess the sensitivity, specificity and cross-reactivity of the CT consensus primer/probe sets.
• NG DNA was similarly prepared and used to assess the sensitivity, specificity and cross-reactivity of the CT consensus primer/probe sets.
• sample DNA In negative control samples (“Neg”), an equal volume of diluent was substituted for sample DNA. Various concentrations of sample DNA of a known concentration were included as positive control (“0.12 ⁇ ”, “1 ⁇ ”, “10 ⁇ ”).
• An internal control (IC) target polynucleotide was designed as positive control for amplification, for use with CT and NG primers.
• the sequence targeted for amplification was complementary to a CT primer at one end and a NG primer at the other end, such that in the presence of both primers the control sequence would be amplified under amplifying conditions.
• the polynucleotide was designed to yield an amplicon complementary to a control probe, for example a molecular beacon probe, but not complementary to the probes in use for the detection of either CT or NG.
• This control target polynucleotide was prepared using methods well known in the art.
• CT DNA was prepared as described in Example 2.
• Primer/probe set 7 was evaluated (i.e., SEQ ID NOs: 1 and 4 were used as primers and SEQ ID NO: 7 as the probe). All sequences were prepared as described in Example 1.
• Dilutions of the isolated CT DNA were PCR amplified and detected using primer/probe set 7.
• PCR was performed in 1 ⁇ GeneAmp® PCR Gold Buffer, 0.25 mM EDTA and 0.125 mM EGTA.
• Amplitaq Gold® DNA polymerase was used at a concentration of 10 units/reaction, with dNTPs (dATP, dGTP, dTTP and dCTP) present at a final concentration of 0.20 mM each.
• a final concentration of 8 mM MgCl 2 was used in a total reaction volume of 0.1 ml.
• Sample volumes were 50 ⁇ l, containing none or 10 1 -10 6 Chlamydial elementary bodies (EB)/reaction.
• Primers were used at a concentration of 200-400 nM each, and beacon probes at a concentration of 100 nM each.
• An internal control (IC) target sequence and control probe were present at a final concentration of 500 copies/reaction and 100 nM respectively
• Reaction mixtures were amplified in a Perkin-Elmer Thermal Cycler. Reaction mixtures were first incubated at 95° C. for 9.5 minutes. PCR amplification was then accomplished in 45 cycles with each cycle being 95° C. for 30 seconds then 59° C. for 60 seconds. After the reaction mixtures were thermal cycled, the mixtures were maintained at 95 ° C. for 3 minutes and then lowered to 25° C. for 10 minutes for probe hybridization. Reaction products were detected on a robotic microplate fluorescence reader using the following emission maxima and band widths (in nm):
• this Example shows good target response over a range of 10 to 10 6 EB/reaction.
• CT and NG DNA were prepared as in Example 2, and the samples prepared as outlined below.
• the CT primer/probe set #7 SEQ ID NOs: 1 and 4 as primers and SEQ ID NO: 7 as probe
• NG primer/probe set #12 SEQ ID NOs: 8 and 9 as primers and SEQ ID NO: 12 as probe
• the samples included 2 cross-reactivity panels (samples 1-58 and samples 1B-23B) as well as negative (Neg) and positive controls (0.12 ⁇ , 1 ⁇ and 10 ⁇ ).
• Each reaction mixture comprised a final concentration of 0.40 uM CT forward primer (SEQ ID NO: 1), 0.20 uM CT reverse primer (SEQ ID NO: 4), 0.10 uM CT beacon probe (SEQ ID NO: 7), 0.20 uM NG forward primer (SEQ ID NO: 8), 0.20 uM NG reverse primer (SEQ ID NO: 9), 0.10 uM NG beacon probe (SEQ ID NO: 12), 5 units of Amplitaq DNA Polymerase, 1 mM dNTPs, and 8 mM MgCl 2 in 1 ⁇ PCR Buffer II. 100 ul reactions were cycled 45 times through 95° C./30 sec and 59° C./1 min for DNA amplification, followed by 1 cycle at 95° C./3 min and 25° C./10 min for final denaturation and
• detection of CT was not altered by the inclusion of the NG primer/probe set with the CT primer/probe set in the CT reaction mixtures, with sensitivity remaining at approximately 10 7 organisms per reaction.
• detection of NG was not altered by the inclusion of the CT primer/probe set with the NG primer/probe set in the NG reaction mixtures, with sensitivity remaining at approximately 10 7 organisms per reaction.
• the NG assay reagents detected NG, but did not detect any of over 50 other non-sexually transmitted (non-STD) strains of Neisseria , nor 3 strains of Chlamydia ( trachomatis, pneumoniae , and psittaci ).
• CT primer/probe set #7 and NG primer/probe set #12 were prepared as described in Example 1, and the CT and NG control DNA were prepared as in Example 2.
• the CT serovar samples were present in the reaction mixtures at final concentrations in the range of 10-1000 molecules per reaction.
• Each reaction mixture comprised a final concentration of 0.40 uM CT forward primer (SEQ ID NO: 1), 0.20 uM CT reverse primer (SEQ ID NO: 4), 0.10 uM CT beacon probe (SEQ ID NO: 7), 0.45 uM NG forward primer (SEQ ID NO: 8), 0.25 uM NG reverse primer (SEQ ID NO: 9), 0.10 uM NG beacon probe (SEQ ID NO: 12), 3 units of Amplitaq DNA Polymerase, 1 mM dNTPs, and 8 mM MgCl 2 in 1 ⁇ PCR Buffer II. 100 ul reactions were cycled 45 times through 95° C./30 sec and 59° C./1 min for DNA amplification, followed by 1 cycle at 95° C./3 min and 25° C./10 min for final denaturation and probe annealing.
• a dilution panel of 7 NG auxotypes was tested in reactions that included primer/probe sets for both CT and NG, and suitable negative (Neg) and positive ( ⁇ and 0.12 ⁇ ) control samples.
• the CT primer/probe set #7 and NG primer/probe set #12 were prepared as described in Example 1, and the CT and NG control DNA were prepared as in Example 2.
• the NG auxotype samples were present in the reaction mixtures at final concentrations in the range of 10-1000 molecules per reaction.
• Each reaction mixture comprised a final concentration of 0.40 uM CT forward primer (SEQ ID NO: 1), 0.20 uM CT reverse primer (SEQ ID NO: 4), 0.10 uM CT beacon probe (SEQ ID NO: 7), 0.20 uM NG forward primer (SEQ ID NO: 8), 0.20 uM NG reverse primer (SEQ ID NO: 9), 0.10 uM NG beacon probe (SEQ ID NO: 12), 3 units of Amplitaq DNA Polymerase, 1 mM dNTPs, and 8 mM MgCl 2 in 1 ⁇ PCR Buffer II. Reactions were cycled 45 times through 95° C./30 sec and 59° C./1 min for DNA amplification, followed by 1 cycle at 95° C./3 min and 25° C./10 min for final denaturation and probe annealing.

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